Pathology Feature Will iam R. Hart, M.D.

Section Editor

Ultrastructural and immunomorphologic study of perinuclear filaments in Merkel cell tumors1

James T . McMahon, Ph.D. Raymond R. Tubbs, D.O. Wilma F. Bergfeld, M.D. Ronald G. Wheeland, M.D Philip L. Bailin, M.D.

Two cases of cutaneous small-cell tumor in patients without overt lung disease are presented. Histologic and ultrastructural findings support the diagnosis of Merkel cell tumor. Immunohistochemistry and immu-noelectron microscopy demonstrate distinctive peri-nuclear keratin filaments that may be of histologic significance in separating a Merkel cell tumor from other nonendocrine tumors. A possible mechanism for the formation of perinuclear filamentous whorls is discussed.

Index terms: Pathology features • Skin neo-plasms

Cleve Clin Q 52:103-110, Spring 1985

The Merkel cell was first described in 1875,1

and has since been described as a distinct type of neurotactile cell acting as a mechanoreceptor and transducer in vertebrate skin.2 Its structure, lo-cation, and association with nerve endings have implied a sensory function, while the ultrastruc-

1 Departments of Pathology (J.T.M., R .R .T . ) and Dermatology (W.F.B., R.G.W., P.L.B.), T h e Cleveland Clinic Foundation. Sub-mitted for publication Oct 1984; revision accepted Dec 1984.

0009-8787/85/01/0103/08/$3.00/0

Copyright © 1985, The Cleveland Clinic Foundation

tural finding of dense-core granules has sug-gested the presence of a chemical transmitter substance.3-5 The histochemical finding of metenkephalin3 and neuron-specific enolase (NSE)4 in Merkel cells has helped affirm its func-tion as a receptor cell, although recent electro-physiologic studies have challenged this concept.6

In 1972, Toker7 described a primary carcinoma of the skin, composed of small cells in rosette-like nests and anastomosing trabeculae. Electron-microscopic studies of similar skin tumors showed intracytoplasmic neurosecretory granules typical of those seen in Merkel cells.8 Descriptions of other primary skin tumors containing neuroen-docrine cellular similarities5,9-13 have suggested a link between small-cell tumors of the skin and the Merkel cell.

While histologic silver stains and immunohis-tochemical methods have generally been unreli-able indicators of Merkel cell tumors,5'9-11'1314

electron microscopy has demonstrated the con-sistent presence of perinuclear filamentous whorls that may be useful in the differential diagnosis of cutaneous small-cell tumors.13

We present two cases of cutaneous small-cell carcinoma in patients without lung tumors. Elec-tron microscopy and immunohistology are pre-sented as potentially useful approaches to the

103

1 104 Cleveland Clinic Quarter ly Vo l . 52, No . 1

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di f ferent iat ion o f these neoplasms f r o m nonen-docr ine tumors. T h e perinuclear f i lamentous whorls found in Merke l cell tumors are also de-scribed.

Materials and methods

Surgical and biopsy specimens f r om 2 patients with nonulcerating skin masses were prepared f o r l ight-microscopic, immunohistologic, elec-tron-microscopic, and immunoelectron-micro-scopic studies. Histologic sections o f formalin-f ixed tissue were stained with hematoxyl in and eosin, Fontana-Masson, and Grimel ius stains.

Immunohisto log ic studies were done on for-malin-f ixed, para f f in-embedded tissue sections, using rabbit antibodies to keratin, somatostatin, calcitonin, and N S E (Dako) . Sections were incu-bated with biotinylated af f inity-puri f ied goat anti-rabbit I g G and p r e f o rmed avidin-biotiny-lated complexes.1 ' A color reaction product was deve loped with 3-amino, 9-ethylcarbazole ( A E C ) and hydrogen perox ide and counterstained with hematoxyl in. Appropr ia te positive and negative controls were done.

Material f o r electron microscopy was made available through either pr imary gluteraldehyde f ixation or electron-microscopic processing o f formal in- f ixed materials, each with postf ixation in osmium tetrox ide and embedment in Spurr resin. T h i n sections were stained with uranyl acetate and lead citrate.

Iminunoelectron-microscopic studies were per-f o rmed on formal in- f ixed sections, cut on a Vi-

bratome at 40 /u. Sections were immersed in antikeratin polyclonal antibodies and subse-quently treated in a sequence o f solutions similar to those used for l ight-microscopic antibody lo-calization. Diaminobenzadine, however , was sub-stituted f o r A E C to f o rm osmiophil ic reaction products that could be visualized under the elec-tron microscope fo l l owing osmication, embed-ment, and thin sectioning. Sections produced in this manner were v i ewed unstained so as to easily distinguish between electron-dense antibody binding sites and other nonreact ive cellular com-ponents.

Case reports Case 1. A 77-year-old man had been treated with super-

ficial radiation for acne as an adolescent. Beginning at the age of 43 years and continuing for 29 years, numerous facial basal cell carcinomas developed, which were treated by excision. At the age of 71, lie underwent right hemicolec-tomy for adenocarcinoma. On admission, he had a translu-cent, reddish-brown 4.0-mni papule adjacent to the ver-milion border of the lower lip (Fig. 1), which was diagnosed on biopsy as a Merkel cell tumor and was resected as a 5.0-cm nodular tumor located just beneath the lip mucosa. Chest radiographs at that time showed no active lesion. The pa-tient subsequently received cobalt treatment to cervical lymph nodes for what was described as Merkel cell tumor metastases. The patient was readmitted several months later with abdominal pain and a 11.4-kg (25-pound) weight loss and was found to have an enlarged liver with a metastatic neoplasm demonstrated by computed tomography. The pa-tient declined treatment, did not undergo liver biopsy, and died shortly thereafter. Autopsy was not performed.

Case 2. A 67-year-old woman had a right axillary mass measuring 5.9 cm in greatest dimension. It was diagnosed as a Merkel cell tumor and removed by wide excision. A second Merkel cell tumor appeared at the same site within a year, at which time clinical evaluation demonstrated dis-tant metastases to the liver. Chest radiographs were unre-markable. The patient received a course of chemotherapy, but died within 18 months of the original diagnosis. Autopsy was not performed.

Results

O n light microscopy (Fig. 2), both tumors were seen to be conf ined to the upper dermis. T h e y were composed o f cells with a small amount o f eosinophilic cytoplasm div ided into clusters or nests by a fine fibrovascular network. Cells were also organized into large epithel ioid sheets, or occasionally had infi ltrated between collagen bundles as trabecular extensions into the dermis. Individual cells were o f uni form size ( 1 5 - 2 0 tx in diameter) . Nuclei were general ly round to oval and exhibited finely granular chromatin with fre-quent invagination o f the nuclear membrane.

Spring 1985 Perinuclear filaments in Mcrkcl cell tumors 105

Fig. 2. Dermal and subcutaneous small-cell tumor demonstrates a predominantly solid growth pattern (hematoxylin and eosin stain, X325).

Fig. 3. Tumor cells with spherical perinuclear bodies containing immunoreactive keratin (arrows) (avidin-biotinylated peroxidase technique with amirioethylcarbazole as chromogen; hematoxylin counterstain, x 1,300).

1 106 Cleveland Clinic Quarter ly Vol . 52, N o . 1

Fig. 4. Tumor cells contain few organelles ultrastructurally. Distinctive perinuclear filamentous whorls can be identified (uranyl acetate and lead citrate, X5.600).

Fig. 5. Filamentous whorls shown here contain 10-12-nm intermediate filaments (uranyl acetate and lead citrate, X20,000).

Spring 1985 Perinuclear fi laments in Mcrkcl cell tumors 107

Fig. 6. Inimunoelectron microscopic staining of tumor cells demonstrates antikeratin binding to perinuclear filamentous whorls (avidin-biotinylated peroxidase technique with diaminobenzidine as chromogen; unstained, x 16,000).

Cells had a high mitotic rate, with 3 - 5 division figures per high-power field. Special stains, in-cluding Fontana-Masson and Grimelius, were negative. Immunohistologic stains for calcitonin, somatostatin, and NSE were also negative.

Immunolocalization of keratin revealed dis-crete, intracytoplasmic staining o f spherical, per-inuclear bodies, usually in proximity with nuclear clefts or invaginations (Fig. 3). These immuno-labeled bodies were easily over looked with casual light microscopy, but were distinctive and con-sistent on careful examination. T h e r e were one or two bodies per cell with either a patchy or diffuse distribution.

Electron microscopy demonstrated small, round to polygonal cells with poorly formed des-mosomal junctions. Nuclei were variably clefted and round to oval with dispersed chromatin and small eccentric nucleoli. T h e scanty cytoplasm contained sparse organelles and was generally unimpressive, except for the uniform presence of perinuclear bodies (3 /u in diameter), composed of whorls o f intermediate-sized filaments (10-12 nm in diameter) frequently found within the nuclear cleft (Figs. 4 and 5). T h e bodies were

non-membrane-bound, but were sharply demar-cated f rom the rest o f the cytoplasm, and could be seen readily in low-power fields as low-density regions immediately adjacent to the nuclei (Fig. 4). Inimunoelectron microscopy conf i rmed the keratin content o f the cytoplasmic filaments (Fig. 6). Cell membranes were generally smooth, but occasional dendritic processes contained scant to abundant numbers of dense-core neurosecretory granules (150-200 nm in diameter) with primary deposition near the cell periphery (Fig. 7).

Discussion Merkel cells in the skin of humans and other

animals are closely associated with nerves and produce met-enkephalin^ and NSE,4 thereby sup-porting the concept that Merkel cells may act as transducers in neurotactile stimulation of mechanoreceptor cells. It has also been hypoth-esized that Merkel cells are part o f the A P U D cell system proposed by Pearse" ' as a classification o f hormone-producing neuroectodermally de-rived cells that have Amine content, amine Pre-cursor i/ptake, and amino acid Decarboxylase activity (APUD). Ultrastructurally, Merkel cells

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have dense-core granules, further supporting the hypothesis o f a neurosecretory function.3,4

Merkel cell tumors are a rare but well-known entity described by various names (trabecular carcinoma,7 primary small cell carcinoma o f the skin,1' neuroendocrine carcinoma of the skin,'1

cutaneous APUDoma , 1 8 and small cell neuroepi-thelial tumors o f the skin12). It is important to recognize Merkel cell tumors due to their local aggressiveness, tendency to invade regional lymph nodes, and ability to metastasize to distant sites. Histologically, they can resemble metastatic oat cell bronchogenic carcinoma, carcinoid tu-mor, neuroblastoma, sweat gland carcinoma, or lymphoma. On light microscopy, Merkel cell tu-mors can be seen as groups o f small cells growing in expansive sheets or forming organoid or tra-becular patterns. Pseudoglandular formations and cellular rosettes may be seen along with prominent necrosis. Prominent lymphocytic and plasmacytic infiltrates may be seen peripheral to tumor cell masses and admixing with tumor cells. T h e localization of calcitonin19 and somato-statin14 in such tumors implies A P U D derivation,

but the variability in these immunodemonstra-tions and the inconsistency in silver staining has made histologic classification o f these tumors un-reliable and misleading.

T h e two cases reported here are consistent with other histologic descriptions o f Merkel cell tumors, and as also reported in the litera-ture,̂ r,.«.i-i ja ck p 0 s i t j v e staining for both silver stain and immunolocalization o f calcitonin, so-matostatin, and NSE. Presentation of these cases is intended to demonstrate the utility o f electron microscopy and immunohistology in differentiat-ing Merkel cell tumors f rom other nonendocrine skin tumors, even in cases where proliferating tumor cells have lost the potential to produce polypeptide hormones or biogenic amines, or when silver stains are inconclusive.

Recent reports9 " '3 have indicated that peri-nuclear whorls o f filaments, like those described here, are consistently reproducible in Merkel cell tumors and that their ultrastructural identifica-tion may be o f diagnostic importance, especially in excluding nonneuroendocrine skin tumors, which lack such features. Sidhu20 has described

Spring 1985 Perinuclear filaments in Mcrkcl cell tumors 109

round masses of cytoplasmic filaments as indica-tors of embryologie origin in a series of APUD tumors, including small cell carcinomas of the lung and carcinoid tumors from a variety of sites. Similar-appearing filaments have also been re-ported as a common finding in pituitary adeno-mas.21 Our study demonstrates the consistent presence of intracytoplasmic perinuclear whorls of intermediate filaments (10-12 nm) in skin tumor cells that also contain dense-core neurose-cretory granules. The prominence of these fila-ment bodies prompted us to further investigate their composition and to establish their presence at the light-microscopic level.

Polyclonal antibodies to keratin, demonstrated with immunoperoxidase methods, confirmed the keratin content of the filaments at the electron-microscopic level and demonstrated potential usefulness of antikeratin staining of small cell tumors of the skin at the light-microscopic level. The distinctive staining pattern of one or two spherical bodies per cell presaged ultrastruc-turally defined perinuclear whorls of keratin fil-aments and provided histologic evidence suggest-ing a neuroendocrine tumor despite negative sil-ver staining and negative immunostaining for biogenic amines.

The function and genesis of the filamentous whorls in Merkel cell tumors are speculative. They may reflect perturbation of the natural state brought on by neoplastic transformation. Intermediate filaments are normally associated with cytoskeletal maintenance of cell shape and reflect a constancy in the functional state of the cell. The filamentous whorls in Merkel cell tu-mors may reflect continual conformational changes precipitated by a high mitotic rate, pre-cluding cellular commitment to cell shape. The perinuclear pooling of filaments is a possible re-sult of this noncommitment. Goldman22 demon-strated perinuclear spheres of 10-12-nm fila-ments in cultured cells treated with colchicine and suggested that microtubules may help deter-mine the normal distribution of intermediate fil-aments in spread cells. Under the influence of alkaloids, these filaments may collapse and be-come arrested as perinuclear caps. In a rapidly dividing tumor cell, microtubules are continually being assembled and disassembled during the repeated process of mitosis; hence, it is possible that without the sustained influence of formed cytoplasmic microtubules, intermediate filaments may form perinuclear whorls like those seen in

Merkel cell tumors. Why these filamentous whorls seem to appear principally in neuroendo-crine tumors is not clear.

James T. McMahon, Ph.D. Department of Pathology The Cleveland Clinic Foundation 9500 Euclid Ave. Cleveland OH 44106

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eren und beim Menschen. Arch Mikrosk Anat 1875; 11:636-652.

2. Smith KR Jr. The Haarscheibe. J Invest Dermatol 1977; 69:68-74.

3. Hartschuh W, Weihe E, Büchler M, Helmstaedter V, Feurle GE, Forssmann WG. Met-enkephalinlike immunoreactivity in Merkel cells. Cell Tissue Res 1979; 201:343-348.

4. Gu J, Polak JM, Tapia FJ, Marangos PJ, Pearse AGE. Neuron-specific enolase in the Merkel cells of mam-malian skin. The use of specific antibody as a simple and reliable histologic marker. Am J Pathol 1981; 104:63-68.

5. Warner TF, Uno H, Hafez GR, et al. Merkel cells and Merkel cell tumors. Ultrastructure, immunocytochemistry and review of the literature. Cancer 1983; 52:238-245.

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7. Toker C. Trabecular carcinoma of the skin. Arch Dermatol 1972; 105:107-110.

8. Tang C-K, Toker C. Trabecular carcinoma of the skin: an ultrastructural study. Cancer 1978; 42:2311-2321.

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10. Sidhu GS, Feiner H, Flotte TJ, Mullins JD, Schaefler K, Schultenover SJ. Merkel cell neoplasms. Histology, electron microscopy, biology, and histogenesis. Am J Dermatopathol 1980; 2:101-119.

11. Pilotti S, Rilke F, Lombardi L. Neuroendocrine (Merkel cell) carcinoma of the skin. Am J Surg Pathol 1982; 6:243-254.

12. Pollack SV, Goslcn JB. Small-cell neuroepithelial tumor of skin: a Merkel-cell neoplasm? J Dermatol Surg Oncol 1982; 8 :116-122 .

13. Wick MR, Goellner JR, Scheithauer BW, Thomas JR III , Sanchez NP, Schroeter AL . Primary neuroendocrine carci-nomas of the skin (Merkel cell tumors). A clinical, histologic, and ultrastructural study of thirteen cases. Am J Clin Pathol 1983; 79:6-13.

14. Silva EG, Ordonez NG, Lechago J. Immunohistochemical studies in endocrine carcinoma of the skin. Am J Clin Pathol 1984; 81:558-562.

15. Hsu SM, Raine L, Fanger H. A comparative study of the peroxidase-antiperoxidase method and an avidin-biotin com-plex method for studying polypeptide hormones with radio-immunoassay antibodies. Am J Clin Pathol 1981; 75:734-738.

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16. Pearse AGE. Common cytochemical and ultrastructural characteristics of cells producing polypeptide hormones (the A P U D series) and their relevance to thyroid and ultimobran-chial C cells and calcitonin. Proc R Soc Lond (Biol) 1968; 170:71-80.

17. Taxy JB, Ettinger DS, Wharam MD. Primary small cell carcinoma of the skin. Cancer 1980; 46:2308-2311.

18. DeWolf-Peeters C, Marien K, Mebis J, Desmet V. A cuta-neous APUDoma or Merkel cell tumor? A morphologically recognizable tumor with a biological and histological malig-nant aspect in contrast with its clinical behavior. Cancer 1980; 46:1810-1816.

19. JohannessenJV, Gould VE. Neuroendocrine skin carcinoma associated with calcitonon production: a Merkel cell carci-noma? Human Pathol 1980; l l (suppl) :586-589.

20. Sidhu GS. T h e endodermal origin o f digestive and respira-tory tract A P U D cells. Histopathologic evidence and a review of the literature. Am J Pathol 1979; 96:5-20.

21. Horvath E, Kovacs K. Morphogenesis and significance of fibrous bodies in human pituitary adenomas. Virchows Arch [Cell Pathol] 1978; 27:69-78.

22. Goldman RD. The role o f three cytoplasmic fibers in BHK-21 cell motility. I. Microtubules and the effects o f colchi-cine. J Cell Biol 1971; 51:752-762.